Eur J Cardiothorac Surg 2006;29:902-907
© 2006 Elsevier Science NL
Pretreatment with recombined human erythropoietin attenuates ischemia–reperfusion-induced lung injury in rats
Haiwei Wu
a
,
*
,
Binhui Ren
a
,
Jiaquan Zhu
a
,
Guohua Dong
a
,
Biao Xu
a
,
Changtian Wang
a
,
Xiaogang Zheng
b
,
Hua Jing
a
a Department of Cardiothoracic Surgery, Jingling Hospital, Clinical Medicine School of Nanjing University, 305 Zhongshan East Road, Nanjing 210002, China
b Department of Pathology, Jingling Hospital, Clinical Medicine School of Nanjing University, Nanjing, China
Received 29 September 2005;
received in revised form 14 February 2006;
accepted 20 February 2006.
* Corresponding author. Tel.: +86 25 80860075; fax: +86 25 84819984. (Email: wu_haiwei{at}163.com).
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Abstract
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Objective: Based on the findings that erythropoietin (EPO) has been proved to be a multiple functional cytokine to attenuate ischemia–reperfusion (I/R) injury in various organs such as brain, heart, and kidney in animals, this experiment was designed to evaluate the effect of pretreatment with recombined human erythropoietin (rhEPO) on I/R-induced lung injury. Methods: Left lungs of rats underwent 90 min of ischemia and then were reperfused for up to 2 h. Animals were randomly divided into three experimental groups as sham group, I/R group, and rhEPO + I/R group (a single dose of rhEPO was injected intraperitoneally 3000 U/kg 24 h prior to operation). Lung injury was evaluated according to semi-quantitive analysis of microscopic changes, tissue polymorphonuclear neutrophils (PMNs) accumulation (myeloperoxidase (MPO) activity), and pulmonary microvascular permeability (Evan's blue dying method). Peripheral arterial and venous blood samples were obtained for blood–gas analysis after 5 min occlusion of right lung hilus at the end of reperfusion. The serum concentration of tumor necrosis factor (TNF)-
was also measured by the method of enzyme-linked immunosorbent assay. Results: Histological injury scoring revealed significantly lessened lung alveolus edema and neutrophils infiltration in the rhEPO pretreated group compared with I/R group (p
< 0.05). The rhEPO pretreated animals exhibited markedly decreased lung microvascular permeability (p
< 0.05) and myeloperoxidase activity (p
< 0.05). Blood–gas analysis demonstrated that the pretreated animals had significantly ameliorated pulmonary oxygenation function (p
< 0.05). The serum concentration of tumor necrosis factor- in rhEPO pretreated group was markedly decreased compared with that of I/R group (p
< 0.05). Conclusions: Pretreatment with rhEPO appears to attenuate I/R-induced lung injury. This function is partly related with the capacity that rhEPO inhibits the accumulation of polymorphonuclear neutrophils in lung tissue and decreases the systematic expression of tumor necrosis factor-.
Key Words: Erythropoietin • Ischemia–reperfusion injury • Lung
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1. Introduction
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In the past 20 or more years, lung transplantation has been an established treatment for many end-stage lung diseases. Although surgical technique, postoperative management and especially graft preservation methods have been greatly improved within recent years, clinical research has demonstrated that ischemia–reperfusion (I/R) induced lung injury keeps highly up to about 22% of patients after transplantation and remains the main obstacle against successful transplantation [1,2]. Therefore, prevention or attenuation of I/R injury in transplanted lungs might lead to profound effects in those recipient patients and further extend the marginal donor lungs to suitable ones for transplantation.
Recent studies have explored that pharmacologic intervention prior to or at reperfusion rather than during preservation period could preserve function in various organs suffering from I/R-induced injury. Most important to these studies, erythropoietin (EPO), a hematopoietic cytokine produced by the fetal liver and adult kidney in response to hypoxia, has been extended from the classical role of erythroid maturation to one that offers protection against I/R injury in a wide variety of tissues [3]. In rat cardiac I/R injury models, administration of recombined human erythropoietin (rhEPO) before ischemia was associated with the decrease of infract size by inhibiting the expression of nuclear factor 
B and increasing the expression of heat shock protein 70 [4]. A single high dose of rhEPO preischemic treatment has been reported to attenuate lipid peroxidation in experimental liver I/R injury [5]. Preconditioning with erythropoietin protects against subsequent ischemia–reperfusion injury in rat kidney [6]. In addition to these work, it has been reported that rhEPO could attenuate different kinds of lung injury. Rats exposed to hyperoxia exhibited well-maintained alveolar structure and enhanced vascularity when treated with rhEPO [7]. Importantly, rhEPO has been reported to protect the ultrastructure of tracheobronchial epithelia and pulmonary type II epithelia in rats enduring traumatic brain injury [8,9].
In the light of the above-mentioned findings, the present study aims to prove the hypothesis that pretreatment with rhEPO offers pulmonary protective effect against I/R injury in rats.
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2. Materials and methods
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2.1 Animals and drug
Adult male Sprague–Dawley rats weighing 350 ± 20 g were obtained from Chinese Academy of Science Nanjing University Animal Centre. All animals received humane care in compliance with European Convention on Animal Care. The following experimental protocol was approved by the Nanjing University Animal Care and Use Committee. The rats were housed and fed at the Animal Center of Jinling Hospital at least 7 days before surgery for accustom to the environment. The rhEPO was purchased from Shenyang Sunshine Pharmaceutical Co., Ltd. (Shenyang, China).
2.2 Surgical procedures
The rats were anesthetized by intraperitoneal injection of pentobarbital 35 mg/kg body weight and then 0.2 mg of atropine was administered intramuscularly. The animals were placed supinely, and a 14-gauge cannula was inserted into the trachea through a midline neck incision. Then the rats were ventilated on a positive pressure respirator (Rodent Respirator, TKR-200C, China) with a standardized inspired oxygen content of 60%. The ventilation parameters were set as following, 2 cm H2O positive end expiratory pressure, 10 cm H2O maximal peak pressure, respiratory rate 60/min, and I:E ratio 1:1.5. Cannulas (22 and 20 gauge) were inserted into the left carotid artery and the right jugular vein, respectively. A median thoracotomy with the sternum cleavage was performed to expose left and right lung lobes. At this point, all animals received 50 U of heparin dissolved in 0.5 ml saline via the right jugular vein. Five minutes later, the left pulmonary hilus, including the left main bronchus, artery, and vein was occluded with a non-crushing microvascular clamp at the end of expiratory with the lung deflated. Peak pressure was then decreased to 8 cm H2O, and respiratory rate increased to 80/min. After the ischemia period maintained for 90 min, the clamp was removed and the left lung was ventilated and reperfused up to 2 h with ventilation parameters same as initial. During the whole experiment, the lungs were kept moist with wet pledget containing normal saline covered over the incision, and body temperature was maintained at 36–37 °C with a heating lamp placed above. Right lung hilus was occluded for 5 min at the end of reperfusion, and then blood samples were obtained from the left carotid artery and right jugular vein for blood–gas analysis.
2.3 Experimental groups
Three groups were generally designed as the following:
- • Sham group (n = 16). The animals had a thoracotomy, and endured a mechanical ventilation of 3.5 h.
- • I/R group (n = 16). Two milliliters of saline solution was administrated intraperitoneally 24 h prior to operation. The animals underwent the full experimental protocol including 90 min ischemia followed by 2 h of reperfusion.
- • rhEPO + I/R group (n = 16). rhEPO was administrated 3000 U/kg diluted in 2 ml saline solution by intraperitoneal injection 24 h prior to operation. The following experimental protocol was the same as that of the I/R group.
There was not any procedure-related mortality in any of the groups. In the present study, microvascular permeability index was measured by Evan's blue dying method. To avoid the negative effect of the dye on microscopic structural observation and biochemical assay, each group was divided into two subgroups equally. One subgroup is for microvascular permeability analysis, and the other for microscopic observation and biochemical assay.
2.4 Semi-quantitive analysis of histological changes
Tissue of left lung lower part was fixed in 10% formalin and embedded in paraffin. Tissue was processed into 6-µm thick slides, stained with hematoxylin and eosin. A scoring system described previously, including three hallmarks neutrophils, alveolar edema and interstitial infiltrate was adopted for semi-quantitive histological analysis of lung injury [10,11]. The slides were graded by a pulmonary pathologist who was blind to animal groups and familiar with scoring system. Each slide was given a score of 0–3 based on the amount of the three hallmarks, and the total score ranging from 0 for normal lung to 9 for most injured lung was calculated.
2.5 Lung microvascular permeability by Evan's blue dying method
To quantify pulmonary microvascular dysfunction secondary to I/R injury, the microvascular permeability was determined by Evan's blue dying method. Upon microvascular dysfunction, this technique is more sensitive than lung wet weight/dry weight ratio, and can reflect the pulmonary edema in early phase [12]. Evan's blue solution was prepared in PBS at the concentration of 100 mg/ml. Animals received 30 mg/kg dye via the right jugular vein at the beginning of reperfusion. The lung tissue was obtained at the end of reperfusion, snap frozen in liquid nitrogen and then homogenized in 5 ml of formamide. The homogenate was incubated at 37 °C for 24 h and then centrifuged at 5000 x
g for 30 min. The optical density of the supernatant was measured at 620 nm. The concentration of Evan's blue was determined according to a standard curve and expressed as milligrams of Evan's blue per gram of wet lung weight.
2.6 Blood–gas analysis
Peripheral arterial and venous blood samples were obtained after a 5-min occlusion of right pulmonary hilus, so that it would be a substitute for the one entirely from left pulmonary vein and artery, respectively. The samples were analyzed immediately in a blood–gas analyzer when obtained. We examined the venous O2 partial pressure (P
vO2) and arterial O2 partial pressure (P
aO2) and calculated their difference (DPa–vO2).
2.7 Polymorphonuclear neutrophils (PMNs) accumulation by myeloperoxidase (MPO) activity
The myeloperoxidase activity assay was used to quantitate polymorphonuclear neutrophils accumulation in the lung tissue. The upper lobe of the left lung was weighted and suspended in 0.5% of hexadecyltrimethylammonium bromide buffer containing 50 mmol/l potassium phosphate (pH 6.0). The buffer volume (ml) is 20 times the weight of lung tissue (g). To release MPO from tissue, the samples were homogenized in ice bath at 5000 rpm four times (each time for 15 s), and then frozen at –20 °C and thawed at room temperature four times. The mixture was then centrifuged at 30000 x
g for 15 min at 4 °C and the supernatant was collected. Assay buffer was composed of 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide (pH 6.0). 0.05 ml of supernatant was mixed with 1.45 ml of assay buffer. The absorbance (A) was recorded at 30 and 90 s at 460 nm. MPO activity is defined as following: MPO activity = 
A/lung weight (g).
2.8 Serum concentration of tumor necrosis factor (TNF)- by enzyme-linked immunosorbent assay
Four milliliters of blood was obtained from peripheral artery and centrifuged at 1500 rpm for 15 min at 4 °C. The supernatant was collected and stored in liquid nitrogen for analysis. The content of tumor necrosis factor- was determined by using a rat TNF- ELISA kit (Diaclone, FR) according to the manufacturer's guidelines. Each sample was tested in duplicate and averaged. TNF- concentration was expressed in picogram per milliliter.
2.9 Statistical analysis
All the data were expressed as mean value ± SD. When comparing differences between groups for non-parametric data, Kruskal–Vallis variance test was used. Otherwise, one-way analysis of variance was adopted for comparisons between multiple groups. When analysis of variance showed a significant difference, the post hoc multiple comparison test was applied to demonstrate the differences between groups. The analysis was performed by SPSS 10.0 and p-values of less than 0.05 were accepted as statistically significant.
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3. Results
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3.1 Lung injury score
The lung injury scores of each group in light microscope are summarized in Table 1
. The total injury score of I/R group and rhEPO + I/R group were both significantly (p
< 0.05) higher than those of sham group. When compared between I/R and rhEPO + I/R groups, the total score was significantly lower in rhEPO + I/R group. When divided further to each marker, the difference mainly derives from significant less neutrophils (p
< 0.05) and alveolar edema in rhEPO + I/R group (p
< 0.05), and there was no significant difference of interstitial infiltrate score between the two groups.
3.2 Lung microvascular permeability
The microvascular permeability in I/R group was about four times of that in sham group (75.7 ± 9.4 mg/g vs 19.3 ± 5.1 mg/g, p
< 0.05). When pretreated with rhEPO, the permeability was significantly reduced (56.5 ± 5.3 mg/g vs 75.7 ± 9.4 mg/g, p
< 0.05), and yet significantly higher when compared with that seen in sham group (p
< 0.05; Fig. 1
).
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Fig. 1. Lung microvascular permeability by Evan's blue dying method. The rhEPO pretreated I/R animals showed a 25% reduction of the increase of Evan's blue content compared with I/R group. I/R, ischemia–reperfusion; rhEPO, recombined human erythropoietin. *
p
< 0.05 versus sham group;
p
< 0.05 versus I/R group.
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3.3 Blood–gas analysis
The P
aO2 of I/R group was significantly lower compared with sham group (99.3 ± 5.1 mmHg vs 148.9 ± 6.8 mmHg, p
< 0.05). When animals were pretreated with rhEPO, the P
aO2 was significantly ameliorated compared with untreated animals (118.8 ± 6.0 mmHg vs 99.3 ± 5.1 mmHg, p
< 0.05), and however, remained significantly lower when compared with sham group. There was a significant difference of P
vO2 between I/R group and sham group (50.1 ± 4.0 mmHg vs 45.4 ± 3.8 mmHg, p
< 0.05). The P
vO2 in rhEPO + I/R group exhibited slightly higher but the difference was not significant when compared with sham group. The DPa–vO2 of either I/R group or rhEPO + I/R group was significantly lower than sham group (48.5 ± 6.6 mmHg vs 102.3 ± 9.8 mmHg, p
< 0.05 and 71.1 ± 6.4 mmHg vs 102.3 ± 9.8 mmHg, p
< 0.05). When the animals were pretreated with rhEPO, the DPa–vO2 increased significantly compared with I/R group (p
< 0.05; Fig. 2
).
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Fig. 2. Blood–gas analysis of peripheral arterial and venous blood. The P
aO2 and DPa–vO2 of the ischemic–reperfused animals were significantly lower than those of sham group. The P
aO2 and DPa–vO2 of rhEPO pretreated group are significantly higher when compared with I/R group. P
aO2, O2 partial pressure of arterial blood; P
vO2, O2 partial pressure of venous blood; DPa–vO2, difference between P
aO2 and P
vO2. I/R, ischemia–reperfusion; rhEPO, recombined human erythropoietin. *
p
< 0.05 versus sham group;
p
< 0.05 versus I/R group.
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3.4 MPO activity
The MPO activity in I/R group was about five times of the baseline in sham group (4.54 ± 0.41 U/g vs 0.9 ± 0.25 U/g, p
< 0.05). When pretreated with rhEPO, the MPO activity decreased about 23% (3.51 ± 0.46 U/g vs 4.54 ± 0.41 U/g, p
< 0.05). The difference between the rhEPO + I/R group and sham group was significant too (3.51 ± 0.46 U/g vs 0.9 ± 0.25 U/g, p
< 0.05; Fig. 3
).
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Fig. 3. MPO activity of lung tissue. When pretreated with rhEPO the MPO activity of lung tissue decreased about 23% compared with I/R group. MPO, myeloperoxidase; I/R, ischemia–reperfusion; rhEPO, recombined human erythropoietin. *
p
< 0.05 versus sham group;
p
< 0.05 versus I/R group.
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3.5 Serum concentration of TNF-
The serum base level of TNF- was 13.8 ± 2.0 pg/ml. When the rats suffered from I/R injury, the concentration of this cytokine reached 18 folds of the base level (250.9 ± 6.9 pg/ml vs 13.8 ± 2.0 pg/ml, p
< 0.05). With the precondition of rhEPO, the TNF- concentration significantly decreased about 42% (145.6 ± 8.0 pg/ml vs 250.9 ± 6.9 pg/ml, p
< 0.05; Fig. 4
).
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4. Discussion
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Many researchers have focused their efforts on preventing I/R injury by means of pharmacologic intervention prior to ischemia or at the time of reperfusion. Attractively, recent works have discovered the potential role of EPO as a multiple functional endogenous mediator offering protective effect against I/R injury in various tissues and organs in animal experiments. However, whether administration of EPO could ameliorate lung I/R injury has not yet been investigated previously. In our present study, we provide convincing data proving the hypothesis that pretreatment of rhEPO before ischemia could attenuate reperfusion-induced lung injury as well.
I/R-induced lung injury is frequently characterized by pulmonary blood–gas exchange dysfunction [13]. The difference between alveolus partial O2 pressure and the one of arterial blood (DPA–aO2) is one of the frequently used parameters to evaluate pulmonary oxygenation function. However, this parameter can neither avoid the impact of base level of O2 in peripheral venous blood on the amount of assimilating O2 via pulmonary blood–gas barrier, nor directly reflect the blood–gas exchange function of the single lung endured I/R injury. Taking into account of this, blood samples were obtained from peripheral artery and vein for gas analysis by 5 min occlusion of opposite lung hilus at the end of operation in the present study. By this means, the obtained blood would be substituted for the one alone from left pulmonary artery and vein. We then examined the O2 partial pressure of arterial and venous blood (P
aO2 and P
vO2), and calculated the difference between them (DPa–vO2). Data analysis showed that the P
aO2 and DPa–vO2 in rhEPO pretreated group is significantly higher compared with that in I/R group, which demonstrates that rhEPO precondition can ameliorate pulmonary oxygenation function after reperfusion. Although the statistical results of these two parameters by variance significance analysis are similar, we are reasonable to believe that DPa–vO2 is of more sensitivity when considering the statistically significant increase of P
vO2 in the I/R group. Attentively, there is a significant decrease of P
aO2 and increase of P
vO2 in I/R group compared with sham group, showing the weakened O2 uptake ability of peripheral tissue. This supported the opinion of remote I/R insult derived from the reperfused organs [14].
Neutrophils accumulation may play an important role in the progress of I/R injury. First, neutrophils attach with the injured vascular endothelium and accumulate in the resident vascular bed, subsequently activated by proinflammatory cytokines, and then infiltrate into lung tissue through blood–gas barrier. When infiltrated, more inflammatory mediators are secreted by the activated neutrophils insulting pulmonary cells. The inflammatory cascade amplifies into a systemic response, which overflows into further graft injury. Many reports emphasize infiltration of activated neutrophils as an important factor causing lung injury [15]. In our present work, the combined markers of MPO activity assay and histological scoring were used to evaluate neutrophils accumulation. Although the amount of neutrophils is not fully equal to its activity in tissue, data analysis of neutrophils infiltration score, which showed significant differences between groups, tends to support the MPO statistical findings. It is reasonable to believe that these two markers of impaired neutrophils recruitment imply that the pretreatment of rhEPO does attenuate the neutrophils accumulation responding to I/R injury.
TNF- has been identified one of pivotal proinflammatory cytokines accelerating I/R injury [10]. Direct evidence that TNF- plays a role in the pathogenesis of experimental lung I/R injury has been obtained in animal models in which blocking of the action by anti-TNF- markedly reduced vascular injury and neutrophils accumulation [16]. Thus, to inhibit inflammatory response has been one possible pathway to attenuate I/R injury. rhEPO has been shown to selectively reduce the influx of inflammatory cells and mediators into the region of injury in a rat model of cerebral ischemia when administered as either a pretreatment or post-treatment [17]. In an experimental inflammatory bowel disease, the level of this proinflammatory cytokine is significantly decreased when treated with rhEPO [18]. In the present study, we found the similar result that precondition with rhEPO significantly reduced the expression of TNF- in the progress of lung I/R, suggesting rhEPO's anti-inflammatory efficacy.
Different from the mechanism by which traditional anti-inflammatory cytokines (e.g., IL-10 and IL-13) inhibit TNF- production directly in vitro and in vivo [19,20], rhEPO appears to affect TNF- release in an indirect way. In cerebral ischemia model, rhEPO exhibits inflammation attenuating activity only in the setting of ischemic injury selectively. The anti-inflammation effects of rhEPO do not result from a direct action upon inflammatory cells known to express EPO receptors (EPO-R) by blocking the release of cytokines [17]. In vitro experiments using cocultures of glial and neuronal cells, in which neuronal death is associated with the release of factors that induce TNF- release by glial cells [21], provide convincing evidence that the anti-inflammatory action of rhEPO is secondary to its neuroprotective activity. Alternatively, rhEPO might exert its anti-inflammatory effects by preventing the generation of molecular signals [22,23]. Even though rhEPO has no direct attenuating effect on proinflammatory cytokine production, there is increasing evidence that rhEPO does provide increased resistance of cellular targets to the effects of inflammation. In fact, it is notable that TNF- can directly inhibit the endogenous EPO production either in vivo or in vitro [24,25]. In this way, the inhibition of endogenous EPO by TNF- might contribute to the role of this mediator in the pathogenesis of I/R injury, and partly explain why exogenously administered rhEPO is especially beneficial.
Collectively based on the findings above, it is reasonable to conclude that rhEPO could attenuate I/R-induced lung injury, partly by decreasing the expression of TNF- and inhibiting PMNs accumulation. Therefore, rhEPO may be valuable as a clinical candidate for such disorders. Although the clinical application of rhEPO has been reported associated with subsequent toxicities (such as hypertensive emergencies, vascular thrombosis, pyrexia, vomiting, and paresthesias), years of clinical use in patients with anemia and chronic renal failure have shown that rhEPO is safe and well tolerated, suggesting that EPO can fulfill the role as a potential ideal protective agent [3]. In addition to the attempts to reduce potential toxicity during EPO administration, future strategies also must seek to optimize the timing of EPO administration.
Certainly, there are limitations of the current study. We investigated the serum concentration but not the activity of TNF-, and detailed pathways involved in how rhEPO effects the expression of TNF- was not investigated either. These seem to be a bit rough. But when considering that this is the initial study on the protective effect of rhEPO attenuating lung I/R injury, we are satisfied with the outcome. In other words, more elaborate experiments are necessary for further research on the efficacy and mechanisms. Relying on the fact that EPO receptors have been identified on endothelial cells and that the intact structure and function of microvascular endothelial cells (MVECs) is essential for lung function, we are now researching on the effect of rhEPO on MVECs and the involved signal pathways in primary cultured lung MVECs.
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Acknowledgments
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This research project was supported by the Natural Science Foundation of Jiangsu Province, China (Item number: BK2005431).
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Abstract
1. Introduction
